Epidermal growth factor receptor (EGFR, also ErbBl) is a single-pass transmembrane receptor tyrosine kinase (RTK) whose signaling is essential for key physiological processes, including cellular growth, migration, adhesion, and apoptosis (Yarden, Nat, Rev. Mol. Cell. Biol., 2:127-137, 2001). As is true for most RTKs, the activation of EGFR is tightly regulated by the availability of ligand, often epidermal growth factor (EGF) or transforming growth factor-α (TGF-α). Consequently, the dysregulation of EGFR expression and signaling has been implicated in numerous forms of cancer and is correlated with poor clinical outcome. Over the last 25 years, a great deal of research has been focused on targeting EGFR and attenuating its activity. One class of targeted therapeutics against EGFR is that of monoclonal antibodies (mAbs), which specifically recognize EGFR and obstruct its activation. The first antibody-based EGFR therapeutic to be clinically approved was cetuximab, the chimeric human immunoglobulin G1 (IgG1) form of the murine mAb 225 (Martinelli et al., Clin. Exp. Immunol., 158:1-9, 2009). With a 100-fold greater affinity for EGFR than the native EGF ligand, mAb 225 directly competes with ligand binding to domain III, blocking dimerization and, consequently, receptor activation (Grunwald et al., J. Natl. Cancer Inst., 95:851-867, 2003; Le et al., Cancer Cell, 7:301-311, 2005). Cetuximab (Erbitux®) can also exert effects via alternative mechanisms, including antibody-dependent cellular cytotoxicity (de Bono et al., Br. Med. Bull., 64:227-254, 2002), induction of receptor internalization and degradation (Ennis et al., Cancer Invest., 9:553-562, 1991), induction of G1-phase cell-cycle arrest, enhanced apoptosis (Prewett et al., Clin. Cancer Res., 8:994-1003, 2002; Ciardiello et al., Clin. Cancer Res., 5:909-916, 1999), and inhibition of vascular endothelial growth factor (VEGF), although these effects vary between cell lines. Other monoclonal antibodies targeting the EGFR ligand-binding domain include the FDA-approved panitumumab and several compounds undergoing clinical trials, including matuzumab and hR-3 (Mateo et al., Immunotechnology, 3:71-81, 1997; Sebastian et al., Biochim. Biophys, Acta, 1766:120-139, 2006).
Unfortunately, the approved mAbs have not lived up to their promise in the clinic. The monotherapy objective response rates of cetuximab and panitumumab are just 11% and 8%, respectively, in the treatment of metastatic colorectal cancer (Cunningham et al., N Engl. J. Med., 351:337-345, 2004; Cohenuram, Anticancer Drugs, 18:7-15, 2007; Van Cutsem, et al., J. Clin. Oncol., 25:1658-1664, 2007). These response rates approximately double when the drugs are used in combination with chemotherapeutics, but there is still much opportunity for the improvement of EGFR-targeted antibody therapeutics. The tepid clinical response of cetuximab and panitumumab can be attributed to delivery limitations, acquired resistance, and receptor mutation (Martinelli, Clin. Exp. Immunol., 158:1-9, 2009). Specifically, antibody penetration into solid tumors is limited by transport and catabolism. Also, tumors may develop resistance to mAbs, often through genetic mutation of EGFR. Heterozygous somatic mutations including deletions, insertions, and point mutations have been observed in the EGFR kinase domain in some lung cancer patients (Lynch et al., N. Engl. J. Med., 350:2129-2139, 2004; Paez et al., Science, 304:1497-1500, 2004; Pao et al., Proc. Natl. Acad. Sci. USA, 101:13306-13311, 2004). These mutations strengthen receptor interactions with ATP, amplifying autophosphorylation and boosting cell survival (Tracy et al., Cancer Res., 64:7241-7244, 2004; Sordella et al., Science, 305:1163-1167, 2004). Furthermore, rearrangements within the ErbB1 gene such as large deletions, point mutants, and insertions are also common, particularly in gliomas (Ekstrand et al., Proc. Natl. Acad. Sci. USA, 89:4309-4313, 1992). As many as 20% of glioblastomas express EGFR variants (Ekstrand et al., Cancer Res., 51:2164-2172, 1991; Liu et al., J Mol. Med., 83:917-926, 2005), the most common of which is EGFRvIII, a constituitively active truncation mutant that removes all of domain I and the majority of domain II of the EGFR extracellular domain to lock the receptor in the active conformation (Wong et al., Proc. Natl. Acad. Sci. USA, 89:2965-2969, 1992). Tumors may also exhibit antibody resistance through abnormal expression of the ligand, for instance through autocrine production or through increased spatial accessibility as a result of aberrant colocalization of the receptor and ligand (Tateishi et al., Cancer Res, 50:7077-7080, 1990; Hirai et at, Dis. Esophagus, 11:221-225, 1998). Due to their reliance on ligand competition for efficacy, the current clinically approved antibodies targeting EGFR are ineffective against mutants such as EGFRvIII and tumor cells that dysregulate EGFR ligands. Consequently, there is a dire need for effective EGFR-targeted mAbs that operate through complementary mechanisms to inhibit receptor signaling.